Three genes (GJB2, GJB3, GJB6) encode for gap junction proteins (connexin 26 (Cx26), (Cx30) and (Cx31) respectively), are found to cause dominant and/or recessive hearing loss. How these mutations cause hearing loss remains unknown. While both recessive and dominant mutants have impaired function, the dominant mutants might have additional dominant negative effects on the wild type counterparts or even trans-dominant inhibition effects on each other, as Cx26, Cx30 and Cx31 are all expressed in the cochlea. The loss of gap junction coupling in cochlear cells may affect the homeostasis of potassium ions in the inner ear and cause hearing impairment. To test these hypotheses, cellular and animal models will be generated to analyze the effects of dominant GJB2/Cx26, GJB6/Cx30, and GJB3/Cx31 mutations, with the following three Specific Aims: 1) to determine the effects of dominant GJB2/Cx26, GJB6/Cx30, and GJB3/Cx31 mutations on the assembly, trafficking and function of their cognate mutant protein in gap junction-deficient HeLa cells; 2) to determine the effects of dominant GJB2/Cx26, GJB6/Cx30, and GJB3/Cx31 mutations on HeLa cells that express wild-type human Cx26, Cx30 and Cx31; 3) to generate and analyze animal models of selected dominant GJB2/Cx26 mutations, by expressing these mutants in the mesenchymal cells of the cochlea. The trafficking and functional results of the cellular and animal models will elucidate the fundamental cellular and molecular mechanism of dominant GJB2/Cx26, GJB6/Cx30, and GJB3/Cx31 mutations, and the animal models of selected mutations will serve as a fundamental resource for further study of the causes and treatments of these inherited causes of deafness. The principle investigator proposed this five year supervised research experience and didactic training to serve as a transitional period from a clinician to a clinician scientist. Dr. Scherer, who has performed similar analyses on the effects of GJB1/Cx32 mutations, will mentor her scientific development, along with Dr. Crenshaw, an established investigator in neurobiology and auditory biology. The work will be supported by the collaboration of Dr. Saunders, an established investigator in the neurobiology and physiology of hearing. An external advisory committee of highly regarded expert in connexin and auditory biology will provide additional support and advice. My goal is to become a well trained independent investigator in biomedicine. The excellent academic environment and the tremendous resource at the University of Pennsylvania, as well as the commitment of her institute will maximize her potential to succeed.